Non-N-methyl-D-aspartate (NMDA) glutamate receptor (GluR) channels are the molecular entities that mediate the majority of the fast excitatory synaptic transmissions in the mammalian central nervous system (Mayer et al, J. Physiol., 354:29-53 (1984)). Studies aimed at improving understanding of the properties of synaptic non-NMDA GluR channels by direct application of glutamate is severely limited by poor access in the intact preparation. A potential solution to this problem is the use of "caged" compounds.
A caged compound is an effector molecule whose activity is temporarily masked by the attachment of a photosensitive masking, or caging, group (Kao et al, In: Optical Microscopy: Emerging Methods and Applications, Herman et al, Eds., Academic Press, San Diego, pages 27-85 (1993); and Adams et al, Ann. Rev. Physiol., 55:755-784 (1993)). Cleavage of the caging group by flash photolysis rapidly liberates the fully bioactive molecule to cause a "jump" in the concentration of the effector molecule. This feature, coupled with the fact that photolysis can be achieved with highly focused light beams, means that photorelease of caged molecules can afford excellent spatial and temporal control over reagent delivery to biological preparations.
In situ photorelease of caged glutamate offers a potentially powerful means for studying the properties of synaptic GluR's, their distribution, and for eliciting action potentials from afar in a specifically targeted neuron (Wang et al, Neuron, 15:755-760 (1995); and Katz et al, J. Neurosci. Meth., 54:205-218 (1994)). However, a number of distinctive properties of GluRs present formidable challenges to the design of caged glutamate reagents. The non-NMDA subset of GluRs require&gt;1.0 mM glutamate for full activation, yet&lt;10 .mu.M glutamate can induce significant desensitization in these same GluRs (Trussell et al, Neuron, 3:209-218 (1989); and Tang et al, Science, 243:1474-1477 (1989)). Furthermore, 10 .mu.M glutamate is sufficient to activate the NMDA subset of GluRs (Mayer et al, supra). An ideal caged glutamate should, therefore, give high yield of free glutamate on photolysis, and should have minimal pre-photolysis activity and high chemical stability. Moreover, because entry into desensitization occurs on the several-millisecond time scale, photorelease must be sufficiently rapid to outpace desensitization. Although there has been considerable effort to perfect a caged glutamate (Wilcox et al, J. Org. Chem., 55:1585-1589 (1990); Corrie et al, J. Physiol., 465:1-8 (1993); Wieboldt et al, Proc. Natl. Acad. Sci., USA, 91:8752-8756 (1994a); and Gee et al, J. Org. Chem., 61:1228-1283 (1996)), no caged glutamate to date has fully satisfied all of these criteria. Some show high chemical stability, but very slow photorelease kinetics (Corrie et al, supra), while others uncage rapidly, but either possess significant pre-photolysis activity or are sufficiently labile as to release glutamate slowly even in the absence of light (Wieboldt et al, supra (1994a); and Gee et al, supra (1996)).
More specifically, the currently most useful caged glutamate, .gamma.-O-(.alpha.-carboxy-2-nitrobenzyl)-glutamate (.gamma.-CNB-Glu), developed by Hess and colleagues (Wieboldt et al, supra (1994a)), exhibits a photorelease rate and quantum yield that are sufficiently high to suit many biological applications. However, in experiments where a caged glutamate is used at high concentration and/or for extended periods in aqueous solution near neutral pH, significant pre-photolysis bioactivity is evident. The pre-photolysis activity of .gamma.-CNB-Glu, which increases with time near neutral pH, is most likely due to the presence of the benzylic carboxylate in the CNB cage, which can catalyze the spontaneous hydrolysis of the ester linkage between the CNB cage and the .gamma.-carboxyl group of the glutamate side-chain to liberate free glutamate. The "neighboring-group-participation" mechanism (Ritchie, Physical Organic Chemistry, Marcel Dekker, New York, pages 80-89 (1975)) for this process is shown in FIG. 1.
As shown in FIG. 1, the benzylic carboxylate first attacks the ester linkage to the .gamma.-carboxyl of glutamate to generate an anhydride. The anhydride is then hydrolyzed by water to yield the intact CNB cage and free glutamate. Such a mechanism involving participation by the neighboring carboxylate on CNB is consistent with the finding herein that generation of pre-photolysis activity increases with time near neutral pH (see FIG. 10B), but is inhibited at rather low pH, because at low pH, the critical carboxylate would become protonated, and thus lose its ability to engage in nucleophilic attack on the ester linkage.
Catalysis by a neighboring carboxylate similarly rationalizes why photorelease of the glutamate .gamma.-carboxyl from the CNB cage is more than 3.5 orders of magnitude faster than from the 1-(2-nitrophenyl)ethyl (NPE) cage, which lacks the benzylic carboxyl group (Wieboldt et al, supra (1994a)) (see FIG. 2).
In designing the compounds of the present invention, retaining the kinetic advantage of the benzylic carboxylate in accelerating photorelease, while eliminating the disadvantage of the same benzylic carboxylate in catalyzing spontaneous hydrolysis of the caged compound to give free glutamate in the absence of light, was sought.
With these concerns in mind, a new caged glutamate has been synthesized and evaluated in the present invention that offers a reasonable compromise in achieving good photolysis yield, rapid kinetics, low intrinsic activity, and chemical stability.
In particular, in N-[2-[2-nitrophenyl]-2-oxycarbonyl acetic acid]-(S)-glutamic acid (N-Nmoc-L-glutamate, or simply, Nmoc-Glu) (FIG. 3), hydrolytic instability is eliminated by attaching the cage to the .alpha.-amino group of glutamate through a carbamate linkage, which is quite resistant to hydrolysis (Greene et al, In: Protective Groups in Organic Synthesis, Wiley-Interscience, New York, pages 309-405 (1991))). Moreover, retention of the benzylic carboxylate in Nmoc-Glu implies that photochemical cleavage, which is otherwise very slow in carbamate-linked cages (Corrie et al, supra), would be significantly accelerated. It was postulated in the present invention that such a design would dramatically increase the hydrolytic stability of Nmoc-Glu relative to .gamma.-CNB-Glu, with only a modest sacrifice in photorelease quantum yield and speed.
Although vulnerability to spontaneous hydrolysis can largely account for the differences in pre-photolysis activity between Nmoc-Glu and .gamma.-CNB-Glu, there may be other contributing factors. For example, attachment of a caging group may not completely abolish the biological activity of an effector molecule (Kao et al supra; discuss the specific case of caged ATP; see also Nichols et al, Pflugers Arch., 415:510-512 (1990); and Ammala et al, Biochim. Biophys. Acta, 1092:347-349 (1991)).
The carbamate linkage between the Nmoc group and the .alpha.-amino group of glutamate has been found in the present invention to effectively eliminate hydrolytic instability, but at the cost of introducing an additional rate-limiting, pH-dependent decarboxylation step, which slows the final release of free glutamate. Glutamate photorelease from Nmoc-Glu thus occurs on the several-millisecond time scale, which is slower than from .gamma.-CNB-Glu. However, it was found in the present invention that glutamate photorelease is still close to two orders of magnitude faster than from N1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate (NPEOC)-caged glutamate (Corrie et al, supra), which also incorporates a carbamate linkage.
The slower kinetics of glutamate release from Nmoc-Glu can be compensated for in the following two ways:
(1) one can accelerate the rate of glutamate generation by increasing the concentration of Nmoc-Glu.
Because the initial photochemical transformation is extremely fast, the photorelease process shown in FIG. 3 can be approximated as: ##STR1##
where H is the hemiacetal intermediate, C is the carbamate of glutamate, G is free glutamate product, and k.sub.1 and k.sub.2 are unimolecular rate constants. For such a sequential reaction scheme, the time-course of glutamate release is given by (Moore, Physical Chemistry, 4th Ed. Prentice-Hall, Englewood Cliffs, N.J., pages 345-346 (1972)): ##EQU1##
where G(t) is the concentration of free glutamate as a function of time, and H.sub.0 is the initial concentration of hemiacetal intermediate produced by photolysis. This integrated rate equation shows that the rate of glutamate generation is directly proportional to the concentration of hemiacetal produced initially by photolysis, which is, in turn, directly proportional to the concentration of caged compound used. Therefore, increasing the concentration of caged glutamate will always result in faster accumulation of free glutamate after photolysis. This compensatory approach to increasing the rate of free glutamate photorelease is possible in the case of Nmoc-Glu, because the pre-photolysis activity of Nmoc-Glu is negligible. Thus, increasing the Nmoc-Glu concentration will always lead to faster accumulation of free glutamate without causing unwanted activation or desensitization of GluRs.
(2) because the rate-limiting decarboxylation to release free glutamate is pH-dependent, one can accelerate photorelease by lowering the pH.
The strategy and method developed for caging the amino acid glutamate in the present invention has been found in the present invention to be readily extended and applied to other amino acids and derivatives thereof.